Serological markers of inflammatory bowel disease phenotype and disease progression

ABSTRACT

Disclosed are novel biomarkers and methods related to diagnostic tests for the detection and characterization of inflammatory bowel diseases, such as Crohn&#39;s disease and ulcerative colitis. In particular, the instant invention relates to novel biomarkers and methods of using such biomarkers to predict disease behavior and severity, to differentiate among disease types, and to optimize selection of treatment options in individuals suspected of having an inflammatory bowel disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/909,153, which was filed on Mar. 30, 2007, the entirety ofwhich is incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

The incidence of the Inflammatory Bowel Diseases (IBD), Crohn's Disease(CD) and Ulcerative Colitis (UC) has increased dramatically over thepast four decades with approximately five million individuals affectedin North America and Europe. While therapeutic options have increasedover the past decade, the ability to classify subtypes of IBDs, predictdisease progression and behavior, and target newer biologic therapies tospecific subgroups of patients has lagged behind. This has led to anempiric step-up approach to therapy, in which increasingly more potentagents are offered until an effective regimen is identified.

The inflammatory bowel diseases are believed to be caused by a complexinteraction between genetic susceptibility and environmental triggersleading to chronic relapsing intestinal inflammation. (Bouma, G. 2003).CD is characterized by discontinuous transmural inflammation which caninvolve any part of the gastrointestinal (GI) tract, although theterminal ileum and proximal colon are most commonly involved. (HanauerS. B. 2006). Conversely, UC is characterized by continuous superficialinflammation limited to the colon, beginning in the rectum and extendingproximally (Hanauer S. B. 2006).

IBDs can be difficult to diagnose, a diagnosis being obtained only afterextensive, costly, and often invasive procedures have been performed. Aninitial diagnosis, made on the basis of medical history and physicalexamination, is generally confirmed via imaging of the intestines andlaboratory culture tests to rule out bacterial, viral and parasiticinfections. Colonoscopy may also be used to image the intestines andcolon, such that the entire intestines can be evaluated to distinguishbetween IBDs on the basis of the location of ulcerations. (Crohn'sdisease affects some areas of the intestines and skips over others,while ulcerative colitis is more indiscriminate.) Endoscopy may also beused to biopsy the intestinal tissue, which can be used to identify thedeep inflammation of the bowel that is characteristic of Crohn'sdisease. Further, X-rays (after oral or rectal ingestion of Barium),computed tomography (CT) scan, and magnetic resonance imaging (MRI) maybe helpful in locating fistulas and assisting in the diagnosis. A stoolanalysis (including a test for blood in the stool) is often performed,depending on symptoms, to look for blood and signs of bacterialinfection. Blood and urine tests may be done to check for anemia, highwhite cell counts, or malnutrition—all signs of IBDs. Finally, theconditions may go undiagnosed for years as symptoms usually developgradually and less than all of the intestines may be involved. As such,while there is currently no reliable biochemical test available for IBDor to distinguish subtypes of the disease, such a test would be highlyvaluable in curtailing cost and the physical discomfort endured byindividuals in obtaining an IBD diagnosis.

With current diagnostic approaches, approximately 60% of IBD patientsare classified as CD, 30% as UC, and 10% as indeterminate colitis (IC)(Kugathasan S. 2003). Patients with IC have intestinal inflammationlimited to the colon, without specific endoscopic or histologicalfeatures diagnostic of either CD or UC. A diagnosis of IC limits theability to predict clinical disease behavior and outcome followingsurgery, and prevents patients with refractory disease from enteringinto clinical trials of new agents.

IBD causes substantial morbidity including frequent hospitalizations andsurgeries, and longstanding disease is complicated by cancer (Hanauer S.B. 2006). It is likely that there are several immunogenetic sub-types ofIBD, with CD and UC representing the broadest clinical classifications(Dubinsky M. C. 2006). Current evidence suggests that CD results whenmucosal tolerance to the enteric flora is lost (Bouma G. 2003, Lodes M.J. 2004). CARD15/NOD2, an intra-cellular sensor for the bacterialproduct muramyl dipeptide, is expressed in intestinal epithelial cells(IEC) and macrophages and participates in anti-microbial defenses (BoumaG. 2003, Kobayashi K. S. 2005). Loss of function mutations in NOD2increases susceptibility to CD primarily involving the ileum (Tomer G.2003).

A variety of defects in neutrophil function have also been described inCD (Korzenik J. R. 2000). These primary defects in mucosal innateimmunity may then lead to activation of intestinal antigen presentingcells (APG) and expansion of T_(eff) reactive to the host flora (BoumaG. 2003, Lodes M. J. 2004). The immunogenetic basis for differences inileal versus colonic involvement in CD is not known, although mountingevidence suggests that defects in innate immunity and loss of toleranceto bacterial antigens are predominately associated with small boweldisease. Mutations in CARD15 are associated with ileal disease andgrowth failure in children, while seroreactivity to microorganismcomponents (ASCA, OmpC, CBir1, and I2 antibodies) is associated withsmall bowel, stricturing disease (Tomer G. 2003), (Mow W. S. 2004). Thisdoes not appear to be the case for UC, in which defects in epithelialrepair and xenobiotic detoxification are implicated (Dignass A. U.2004). The introduction of biologic therapies targeting specificcomponents of the mucosal immune response has improved outcomes forpatients with IBD. However, sustained remissions have not been observedin more than fifty percent of individuals with any new biologic agent.

Current Therapies for Inflammatory Bowel Diseases

Current options for induction of remission in IBD include mesalamine,corticosteroids, methotrexate, and infliximab. Options for maintenanceof remission include mesalamine, the immunomodulators6-mercaptopurine/azathioprine (6-MP/AZA), methotrexate, and infliximab.The most common first line regimen includes induction of remission withprednisone, and maintenance of remission with 6-MP/AZA. In the absenceof a reliable test to predict response to therapy, patients areempirically offered agents for induction and maintenance of remissionlargely based upon disease severity and location. As the effectivenessof any one agent is typically on the order of 50% to 80%, this leads toa substantial number of patients receiving a series of ineffectiveagents, with attendant side effects, before an effective regimen isidentified.

Despite the recent advent of the biologic therapies, corticosteroidsremain the predominant first line choice for induction of remission inmoderate to severe IBD. Typically, 50% of patients will go intoremission and then be able to wean corticosteroids within three months.However, 20% will not respond, and 30% will not be able to discontinuesteroids; these groups are termed steroid refractory (SR) and steroiddependent (SD), respectively.

Inflammatory Bowel Disease Markers for Diagnosis and Progression OfDisease

In recent years, serological markers which largely comprise antibodiesreactive to the host flora have been extensively studied in an attemptto improve IBD classification and prediction of disease progression.However, these markers have not proven effective in definitivelycharacterizing IBD patients as CD or UC, and have not been able topredict with sufficient accuracy which patients are most likely torequire surgery for the condition.

The first serological markers to be described in IBD were theanti-saccharomyces cervisiae antibody (ASCA) and anti-neutrophilcytoplasmic antibody with perinuclear staining (pANCA) (Austin G. L.2006). Subsequently, additional markers including antibodies to theouter membrane porin protein C (OmpC) of E. Coli, to Pseudomonasfluorescens (I2) and to flagellin (CBir1) have been described (DubinskyM. C. 2006). Whether any of these antibodies are pathogenic is notknown, although recent evidence points to bacterial flagellin as acritical antigen in CD (Lodes M. J. 2004). pANCA has been associatedwith UC, while the other markers have been associated with CD. A recentseries of well-characterized adult CD patients demonstrated a frequencyof 37% for OmpC, 52% for I2, 39% for ASCA, and 14% for pANCA (Arnott I.D. 2004). The frequency of pANCA in UC is typically 60% to 70% (AustinG. L. 2006). Multiple studies have been performed to determine whetherlevels of these antibodies can be used to screen for IBD in patientswith suggestive symptoms, and to discriminate CD from UC, therebyreducing the number of cases of IC. However, the sensitivity of pANCAfor UC in recent adult and pediatric series has ranged from 63% to 70%,while the sensitivity of ASCA for CD ranged from 44% to 72% (Linskens R.K. 2002), (Zholudev A. 2004), (Gupta S. K. 2004). Thus, these resultsdemonstrate that these markers are not sufficiently sensitive to be usedas effective screening tools. In terms of specificity, these assaysperform substantially better, and range from 86% to 95% for pANCA for UCand from 82% to 95% for ASCA for CD (Linskens R. K. 2002, Zholudev A.2004, Gupta S. K. 2004). From 10% to 18% of CD patients will be positivefor pANCA, these typically have Crohn's colitis (Zholudev A. 2004). Incohorts of patients with a high prevalence of IBD (42% to 68%), this hasled to a reported positive predictive value (PPV) of 90% to 96% for bothCD and UC, and a negative predictive value (NPV) of 50% to 80% (AustinG. L. 2006, Gupta S. K. 2004). Therefore, as recently reviewed by Austinet al., these tests perform best when used in a patient population witha high pre-test probability of having IBD (Austin G. L. 2006). As such,there is a need for improved markers having improved sensitivity andselectivity that can be used as effective screening tools to diagnoseand differentiate inflammatory bowel diseases.

IBD patients further have variable risk of needing surgery to treat thedisease. Current standard clinical approaches are not able to furtherreduce the number of diagnoses of IC, or to predict which patients willbe most likely to progress to surgery. From about 30% to 50% of IBDpatients will fail medical therapy and progress to surgery within 3 to10 years of diagnosis (Austin G. L. 2006). Most CD patients initiallypresent with inflammatory disease (80%), and over the next 10 years mayprogress to stricturing and/or penetrating behavior, categorized asB2/B3-type disease. (Hanauer S. B. 2006). Patients withstricturing/penetrating disease then frequently require surgery.

Several groups have performed studies to determine whether IBD serologycan predict disease progression and the need for surgery (Mow W. S.2004). Consistently, high titer ASCA+ CD patients have been more likelyto have fibrostenosing small bowel disease and require ileocecalresection (36% vs. 13% in one recent pediatric series) (Arnott I. D.2004). Conversely, pANCA+ CD patients are more likely to have colonicdisease and not require resection. Recent reports demonstrated that thecombined magnitude of reactivity to microbial components (ASCA, OmpC,and I2) predicted a phenotype of more severe small bowl CD withprogression to surgery in adult patients (Arnott I. D. 2004). However,the magnitude of the serological response did not predict the need for6-MP or infliximab. Importantly, the association was also independent ofthe CARD15 genotype, which has also been associated with small bowel CDand progression to surgery (Arnott I. D. 2004, Walker L. K. 2004).

Recent reports in pediatrics have demonstrated very similar predictivecharacteristics. High titer ASCA+ CD patients were more likely torequire surgery sooner than ASCA− patients (Amre D. K. 2006). In a studymeasuring ASCA, OmpC, I2, and CBir1 reactivity, the presence andmagnitude of response of these was associated with a higher rate ofprogression to a stricturing or penetrating complication over the first18 months following diagnosis (Dubinsky M. C. 2006). Potentially thissub-group of CD patients would benefit from earlier institution oftargeted biologic therapies more likely to prevent disease progression.In this regard, Mow et al have recently reported a trend towardsimproved responses to antibiotics in CD patients sero-reactive to OmpCand/or I2, and decreased responses to a steroid preparation, budesonide(Mow W. S. 2004).

A limitation of this approach is the substantial number of patients whoexperience disease progression and require surgery who are sero-negativefor these markers. For example, in the Arnott report, 96 CD patientswere negative for ASCA or OmpC/I12, and fifty percent of thoseexperienced disease progression and required surgery (Arnott I. D.2004). This compared to 40 CD patients who were sero-positive, withrates of disease progression and surgery of 90% and 75%, respectively(Arnott I. D. 2004). Therefore, additional approaches are needed tocomplement current serological assays in defining clinically importantsub-types of IBD with respect to disease behavior and progression.

Current biomarkers have failed to be effectively used to providediagnostic tests for classifying, characterizing or predicting theoutcome of inflammatory bowel diseases. In particular, currentbiomarkers have not been efficacious in distinguishing among the varioussubtypes of inflammatory bowel diseases. Accordingly, there is a needfor biomarkers that can form the basis for diagnostic tools that caneffectively classify, characterize and predict the severity ofinflammatory bowel disease

There is further the need for biomarkers that can be used to predictdisease progression, including the likelihood that an individual willrequire surgery despite treatment with first line therapies would beinvaluable. Such markers would allow the clinician to identify thesubset of patients at an early stage of disease progression who wouldbenefit from earlier introduction of second or third line therapies, ortherapies particularly effective in treatment of a particular subset ofIBD, such as anti-TNFα (for example, Remicade or Humira) and/or GM-CSF(Leukine) or the like. The instant invention satisfies these needs andprovides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to biological markers and methods of usingbiological markers for the diagnosis and prognosis of inflammatory boweldiseases.

The present invention further relates to biological markers that may beused to predict and characterize disease behavior in individuals havingan inflammatory bowel disease.

The present invention further relates to biological markers that may beused to predict disease progression, such as the likelihood that anindividual having an inflammatory bowel disease, particularly Crohn'sdisease, will require surgery.

The present invention further relates to a method of classifying anindividual with a particular subtype of inflammatory bowel disease,comprising the steps of obtaining a biological sample from an individualsuspected of having an inflammatory bowel disease; determining theconcentration of anti-GM-CSF antibodies in the sample; and correlatingthe concentration of anti-GM-CSF antibodies in the sample to knownstandards.

In yet another aspect, the instant invention provides a method ofdiagnosing an individual with a particular subtype of inflammatory boweldisease, comprising the steps of obtaining a biological sample from anindividual suspected of having an inflammatory bowel disease,determining the concentration of anti-GM-C SF relative to neutrophilfunction, IBD phenotype, CARD15 variants or commercial inflammatorybowel disease serology; correlating the results to known standards; anddiagnosing the individual based on these results.

In yet a further aspect, the present invention relates to a method forpredicting the severity of an inflammatory bowel disease in anindividual, particularly whether the individual will require surgery,comprising the steps of obtaining a biological sample from theindividual suspected of having or having an inflammatory bowel disease;determining the concentration of anti-GM-CSF antibodies in the sample;and correlating the concentration of anti-GM-C SF antibodies in thesample to known standards to predict the severity of the inflammatorybowel disease, particularly whether an individual will require surgery.In this aspect of the invention, the method may be used to identify theappropriate therapy for an individual based on the determination ofelevated anti-GM-CSF antibodies.

In yet another aspect, the present invention relates to a method forpredicting the appropriate therapeutic treatment for an inflammatorybowel disease, comprising the steps of obtaining a biological samplefrom an individual suspected of having an inflammatory bowel disease;determining the concentration of anti-GM-CSF antibodies in the sample;and correlating the concentration of anti-GM-CSF antibodies in thesample to known standards to select the appropriate therapeutictreatment for the inflammatory bowel disease, wherein elevatedanti-GM-CSF antibodies indicate that the patient is a candidate fortherapies selected from anti-TNFα therapy, GM-CSF administration, orcombinations thereof.

In another aspect of the present invention, determination of GM-CSFdependent up-regulation of cell surface CD11b may be used as adiagnostic assay to distinguish between UC and CD patients.

The present invention further relates to kits embodying the abovedescribed methods, such as a kit for determining a diagnosis or aprognosis of an individual suspected of having or having an inflammatorybowel disease, which comprises at least a probe specific for GM-CSFwherein the probe is capable of detecting a concentration of anti-GM-CSFantibodies, such that a diagnosis or prognosis of the individual may bemade.

The present invention further relates to immunoassays for thequalitative or quantitative determination, in a sample from anindividual, of anti-GM-CSF antibodies wherein the immunoassay comprisesthe steps of obtaining a sample from an individual suspected of havingor having an inflammatory bowel disease, providing an immobilizedantigen reactive to anti-GM-CSF antibodies, contacting the sample withthe antigen to form an antigen-antibody complex, washing the complex toremove non-specifically bound components, followed by detecting thecaptured anti-GM-CSF antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Anti-GM-CSF Antibody Concentration and IBD Phenotype

FIG. 2. Function of anti GM-CSF antibodies and Neutrophil Phagocytosisin Healthy Individuals and Individuals with Inflammatory Bowel Diseaseand PAP.

FIG. 3. CM-CSF activation of CD11b in Inflammatory Bowel Disease.

DETAILED DESCRIPTION OF THE INVENTION

Current care and clinical trials for patients with IBD is hampered by alack of biomarkers to predict and monitor response to therapy orprogression of disease. Without biomarkers that allow prediction of apatient's responsiveness to current therapies, patients are offeredtherapies via an empiric approach with results in, at best, a 50% rateof sustained remission, while subjecting many patients to unnecessaryside effects and expense, further delaying recovery and improved qualityof life. As such, identification of biomarkers offers the opportunity todefine distinct immunogenetic sub-types of disease which may benefitfrom specific treatment approaches.

For example, the availability of sensitive biomarker would permit thetreating physician to evaluate a patient's likely responsiveness tofirst line or second line therapeutics. The prediction of the patient'slikely response permits the physician to select therapies likely to bemost efficacious for a given individual, avoiding treatment with lessuseful therapies. This, in turn, avoids subjecting the patient tounnecessary side effects and expense, while improving the patient'squality of life and creating opportunities to delay or prevent diseaseprogression.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), provide one skilled in the art with a general guide to manyof the terms used in the present application.

For purposes of the present invention, the following terms are definedbelow.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

A “biological sample” encompasses any sample obtained from a livingsystem or subject. The definition encompasses blood, serum, tissue, andother samples of biological origin that can be collected from a livingsystem, subject or individual. In one embodiment, biological samples areobtained through sampling by minimally invasive or non-invasiveapproaches (e.g., urine collection, stool collection, blood drawing,needle aspiration, and other procedures involving minimal risk,discomfort or effort). Biological samples can be gaseous (e.g., exhaledbreath). Biological samples are often liquid (sometimes referred to as a“biological fluid”). Liquid biological samples include, but are notlimited to, urine, blood, interstitial fluid, edema fluid, saliva,lacrimal fluid, inflammatory exudates, synovial fluid, abscess, empyemaor other infected fluid, cerebrospinal fluid, sweat, pulmonarysecretions (sputum), seminal fluid, feces, bile, intestinal secretions,and others. Biological samples include samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents, solubilization, or enrichment for certain components,such as proteins or polynucleotides. The term “biological sample” alsoencompasses a clinical sample such as serum, plasma, other biologicalfluid, or tissue samples, and also includes cells in culture, cellsupernatants and cell lysates.

As used herein, the term “biomarker” refers to a physical, biochemical,or physiologic measurement from or on the organism that represents atrue or intended mechanistic target of a compound or a mechanistic eventbelieved to be responsible for, or contributing in, a causal manner tothe initiation, progression, severity, pathology, aggressiveness, grade,activity, disability, mortality, morbidity, disease sub-classificationor other underlying pathogenic or pathologic feature of one or morediseases. A biomarker may be the target for monitoring the outcome of atherapeutic intervention (i.e., the functional or structural target of adrug agent). “Biomarker” refers to biochemical processes that areinvolved in, or are believed to be involved in, the etiology orprogression of a disease or disorder. The biochemical process (i.e., theflow of molecules through a targeted metabolic pathway or network) isthe focus of analysis (as disclosed herein) since it is the underlyingchanges of the biochemical process (i.e., molecular flux rates) that maybe the significant or authentic target for treatment or diagnosticmonitoring of the disease or disorder.

The terms “drug,” “pharmaceutically active agent,” “bioactive agent,”“therapeutic agent,” and “active agent” may be used interchangeably andrefer to a substance, such as a chemical compound or complex, that has ameasurable beneficial physiological effect on the body, such as atherapeutic effect in treatment of a disease or disorder, whenadministered in an effective amount. Further, when these terms are used,or when a particular active agent is specifically identified by name orcategory, it is understood that such recitation is intended to includethe active agent per se, as well as pharmaceutically acceptable,pharmacologically active derivatives thereof, or compounds significantlyrelated thereto, including without limitation, salts, pharmaceuticallyacceptable salts, N-oxides, prodrugs, active metabolites, isomers,fragments, analogs, solvates hydrates, radioisotopes, etc.

The phrase “effective amount” refers to that amount of a substance thatproduces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. The effective amount ofsuch substance will vary depending upon the individual and diseasecondition being treated, the weight and age of the individual, theseverity of the disease condition, the manner of administration and thelike, which can readily be determined by one of ordinary skill in theart.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman.

The term “prophylactic” or “therapeutic” treatment is art-recognized andrefers to administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if administeredafter manifestation of the unwanted condition, the treatment istherapeutic (i.e., it is intended to diminish, ameliorate or maintainthe existing unwanted condition or side effects therefrom).

The phrase “therapeutic effect” is art-recognized and refers to a localor systemic effect in animals, particularly mammals, and moreparticularly humans caused by a pharmacologically active substance. Theterm thus means any substance intended for use in the diagnosis, cure,mitigation, treatment or prevention of disease or in the enhancement ofdesirable physical or mental development and/or conditions in an animalor human. The phrase “therapeutically-effective amount” means thatamount of such a substance that produces some desired local or systemiceffect at a reasonable benefit/risk ratio applicable to any treatment.The therapeutically effective amount of such substance will varydepending upon the individual and disease condition being treated, theweight and age of the individual, the severity of the disease condition,the manner of administration and the like, which can readily bedetermined by one of ordinary skill in the art.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) the targeted pathologic condition or disorder.Those in need of treatment include those already with the disorder aswell as those prone to have the disorder or those in whom the disorderis to be prevented.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology and biochemistry,which are within the skill of the art.

All references cited herein are incorporated in their entirety byreference.

The present invention relates to biomarkers that may be used tocharacterize individuals having an inflammatory bowel disease. In oneaspect, the present invention provides novel biomarkers for inflammatorybowel disease that may be used to diagnose subtypes of the disease. Inparticular, the biomarkers as disclosed herein may be used tocharacterize inflammatory bowel disease behavior and progression. In yetanother aspect, the invention provides novel biomarkers that may be usedto predict the likelihood that an individual diagnosed with aninflammatory bowel disease will require surgical intervention.

Further, the biomarkers and methods disclosed herein may be used in thetargeted care of individuals having inflammatory bowel disease. In thisaspect of the invention, Crohn's disease patients having a highanti-GM-CSF level are identified as high risk for disease progressionand surgery, and may be offered second or third line therapies earlierin their treatment. For example, Crohn's disease patients havingelevated anti-GM-CSF antibodies would be candidates for earliertreatment with anti-TNFα therapy (Remicade or Humira), GM-CSF therapy(Leukine) or similar therapies.

A variety of defects in neutrophil function have been described in CD,without a clear etiology (Korzenik J. R. 2000). These have includedreduced phagocytosis, adhesion, chemotaxis, and oxidative burst.Functionally, this has been shown to paradoxically reduce neutrophilaccumulation at sites of acute ileal or rectal injury (Harbord M. W.2006, Mow W. S. 2004). This may in turn promote accumulation ofbacterial products and stimulation of the mucosal adaptive immunesystem. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is aprotein secreted by macrophages that functions as a white blood cellgrowth factor.

GM-CSF and its Role in the Regulation of Myeloid Cell Functions.

GM-CSF is a 23 kD, homodimeric cytokine expressed present in serum andtissues with pleiotropic regulatory effects on the functions of myeloidcells (neutrophils, monocytes, eosinophils, tissue macrophages anddendritic cells). The biological effects of GM-CSF are mediated bycell-surface receptors, comprised of α (CDw116) and β chains (CD131)and, in macrophages and neutrophils include increased phagocytosis,cell-surface levels of CD11b (an adhesion molecule), CD11b, celladhesion, bacterial killing, surfactant catabolism (in alveolarmacrophages) (Trapnell, B. et al., NEJM, 2003, incorporated herein byreference). GM-CSF also primes phagocytosis, oxidative burst, andbactericidal activity in neutrophils. In vitro stimulation with GM-CSFrestores the function of CD neutrophils, and GM-CSF administrationreduces mucosal injury in murine colitis due to trinitrobenzenesulfonicacid (TNBS) or dextran sodium sulfate (DSS) administration (Hanna E.2006), (Fiorucci S. 2006) (Biagioni C. 2006).

While it has been known for some time that GM-CSF could modulate thefunctions of myeloid cells, a critical role for GM-CSF in myeloid cellfunction was discovered by the serendipitous observation that micedeficient in GM-CSF due to gene ablation (GM-CSF knockout mice) developa lung phenotype that is histologically, biochemically,ultrastructurally and physiologically similar to the human disease knownas pulmonary alveolar proteinosis (PAP) (Trapnell, NEJM, 2003). PAP inmice is caused by abrogation of the effects of GM-CSF signaling onmyeloid cell functions, due to ablation of the genes encoding eitherGM-CSF or its receptor. Either genetic manipulation results in areduction in the ability of alveolar macrophages to degrade orcatabolize surfactant. (Shibata, et al., Immunity, 2001, incorporatedherein by reference.) Abrogation of GM-CSF signaling also increases therisk of infection due to loss of the stimulatory effects of GM-CSF onneutrophils (Uchida, et al, NEJM, 2007). The stimulatory effects ofGM-CSF on macrophages and neutrophils are known as ‘priming’, and areknown to augment the antimicrobial functions of these cells, and thus,also augments innate immune host defenses.

GM-CSF Autoantibodies and their Role in Myeloid Cell Functions.

Human primary PAP is a rare disorder in which surfactant withinpulmonary alveoli accumulates, thereby causing respiratory insufficiency(Trapnell et al, NEJM, 2007). Primary PAP is specifically associatedwith high levels of autoantibodies against granulocyte/macrophage-colonystimulating factor (GM-CSF) that neutralize the biological activity ofGM-CSF and are present in vast excess of GM-CSF in vivo. Based onstudies in mice, it is believed that pulmonary GM-CSF stimulates theterminal differentiation of alveolar macrophages including many of theeffects of GM-CSF on alveolar macrophages. A recent study has nowestablished that GMCSF autoantibodies abrogate the effects of GM-CSF onneutrophils in patients with primary PAP (Uchida et al, NEJM, 2007,incorporated herein by reference). Loss of the effects of GM-CSF onneutrophil function caused by GM-CSF autoantibodies provides amechanistic explanation for the morbidity and mortality from infectionin patients with primary PAP.

The molecular basis for the defects in neutrophil function which havebeen described in CD, but not UC, is not known. However, GM-CSF andGM-CSF receptor levels are typically elevated in patients with activeCD, suggesting the role of a serum inhibitory factor. Recent reportsdemonstrating that GM-CSF can correct CD neutrophil function in vitro,and that GM-CSF administration can relieve symptoms and reduceintestinal inflammation in a sub-set of CD patients, have also pointedto a potential role for alterations in GM-CSF bioactivity (Biagioni C.2006, Korzenik J. R. 2005). It has also been determined that very highlevels of anti-GM-CSF auto-antibodies impair neutrophil function andcause the rare lung disease, PAP (Uchida K., et al., Blood 2004,incorporated herein by reference). Neutralizing anti-GM-CSFauto-antibodies have been characterized in patients with the rare lungdisease Pulmonary Alveolar Proteinosis (PAP). (Uchida K. 2004). Higherlevels of anti-GM-CSF inhibit myeloid cell function in patients with therare lung disease PAP. It has been reported that anti-GM-CSFauto-antibodies suppress systemic innate immunity, by preventing GM-CSFpriming of monocyte and neutrophil anti-microbial functions. (Vindi I.2006). These auto-antibodies have been linked to defects in macrophageand neutrophil function and development of disease (Uchida K. 2004).

GM-CSF is currently undergoing phase II clinical trials in CD, withclinical responses reported in approximately fifty percent of patients(Korzenik J. R. 2005). However, a pro-inflammatory role for GM-CSF hasalso been invoked for autoimmune diseases including rheumatoidarthritis, and neutralizing anti-GM-CSF antibodies are also indevelopment as potential therapeutic agents (Krinner E. M. 2006).Moreover, most patients with active CD or UC have increased productionof GM-CSF in the inflamed intestine, and increased local expression ofthe GM-CSF receptors. Endogenous anti-GM-CSF antibodies may regulatelocal GM-CSF bioactivity. It thus is critically important to define thesub-groups of IBD patients who will benefit from therapeutic modulationof GM-CSF bio-activity.

CD11b as a Biomarker of GM-CSF Bioactivity In Vivo.

CD11b is a cell-surface adhesion molecule present on myeloid cells, theconcentration of which increases in response to a wide variety ofinflammatory stimuli. GM-CSF priming increases cell-surface levels ofCD11b on neutrophils, which promotes their adhesion to vascularendothelium, a critical event in recruitment of neutrophils intoinfected tissues. GM-CSF autoantibodies block the GM-CSF-stimulatedincrease in cell surface CD11b levels in PAP patients (Uchida et al,NEJM, 2007). Addition of exogenous GM-CSF to whole blood from a patientcan be used to measure the ability to stimulate this change inneutrophil CD11b levels and is the basis of an assay known as the CD11bstimulation index (Uchida et al, NEJM, 2007, incorporated herein byreference).

The present invention provides novel kits and methods for theclassification, characterization, and prediction of outcome in patientshaving inflammatory bowel diseases. The present invention furtherrelates to kits and methods that may be used to determine the prognosisof an individual having an inflammatory bowel disease, or to determinethe optimal choice of treatment for such individuals.

Anti-GM-CSF Antibody Concentration and IBD Phenotype

In one aspect of the invention, levels of anti-GM-CSF antibody can bemeasured and used to classify individuals having an inflammatory boweldisease. It has been found that IBD patients exhibit a range ofanti-GM-CSF antibody levels, with patients with small bowel CD (CD_(SB))exhibiting high levels, and patients with UC exhibiting low levels,relative to patients with colonic CD and healthy controls. The elevatedanti-GM-CSF levels in patients with CD_(SB) are in the range whichinhibits neutrophil function. Serum levels of anti-GM-CSF independentlyregulate neutrophil function and IBD phenotype, relative to CARD15genotype and current IBD serological markers. Comparing patients withileal or ileo-colonic disease grouped together as small bowel CD(CD_(SB)), and patients with colonic CD grouped as CD_(C), we have foundfor the first time that CD_(SB) patients have increased circulatinglevels of anti-GM-CSF antibodies, while UC patients have decreasedlevels of anti-GM-CSF antibodies, relative to CD_(C) or healthycontrols.

FIG. 1 depicts the relative anti-GM-CSF antibody concentrations in thevarious IBD subgroups compared to healthy control patients. Serumanti-GM-CSF antibody concentrations were determined by ELISA in IBDsub-groups and healthy controls (NL). FIG. 1 depicts anti-GM-CSFantibody concentration in patients with small bowel Crohn's disease(CD_(SB)), patients with Colonic Crohns disease (CD_(C)), and UlcerativeColitis (UC). The differences between groups were compared byKruskal-Wallis with post hoc Bonferroni multiple comparisons test.̂p<0.0001 vs. NL & UC; *p=0.006 vs. CD_(C). The mean value was asindicated for each group.

It was therefore surprisingly discovered that inflammatory bowel diseasepatients having small bowel Crohn's disease (CD_(SB)) had statisticallysignificant increases in anti-GM-CSF antibody concentration as comparedto individuals with colonic Crohn's disease, individuals with ulcerativecolitis, and normal individuals. As such, determination of anti-GM-CSFantibody concentration can be used to distinguish subclasses of patientsdiagnosed with Crohn's disease, providing improved selection oftherapeutic treatments and prediction of disease behavior andprogression.

In fact, mean (±SE) serum anti-GM-CSF concentration in ileal CD_(SB) andileo-colonic CD_(SB) individuals was equal to 2.2 (n=20, and 86,respectively), versus 0.7 mcg/ml (n=51) in CD_(C), 0.7 mcg/ml (n=43) inUC, and 0.4 mcg/ml (n=20) in healthy controls. See Table 1.

Thus, in one aspect of the present invention, a method of classifyingindividuals having an inflammatory bowel disease is provided comprisingthe steps of obtaining a sample from the individual, determining theconcentration of serum anti-GM-CSF concentration in the sample, a mean(±SE) serum anti-GM-CSF concentration of greater than or equal to about1 mcg/ml, or greater than or equal to about 1.5 mcg/ml, or greater thanor equal to about 2.0 mcg/ml, or within the range of about 1.0 mcg/ml to2.5 mcg/ml, indicates that the individual is likely to have ileal orileocolonic Crohn's disease.

Importantly, anti-GM-CSF levels did not change in CD patients who hadachieved clinical remission with anti-TNF therapy (2.4±0.8 mcg/ml vs3.3±1.3 mcg/ml, n=6), indicating that these are a stable feature oftheir disease, and do not simply reflect a response to mucosalinflammation.

The instant invention further relates to a method of modulating IBDphenotype or treating individuals having an IBD using exogenously orendogenously administered GM-CSF.

As shown in FIG. 2, anti-GM-CSF antibodies in the range observed inCD_(SB) can regulate (i.e., neutralize) GM-CSF bioactivity andneutrophil function, thereby creating a functional GM-CSF deficiency inthe individual.

We assessed the potential significance of this finding by quantifyingneutrophil phagocytosis with a sensitive assay. FIG. 2 depicts theresults of this study. FIG. 2 a depicts neutrophil phagocytosis measuredin whole blood. Each dot represents four determinations per individual.αGM-CSF-antibody levels were done in triplicate for each individual.Results demonstrated that neutrophil phagocytic capacity correlatedinversely with αGM-CSF antibody levels, over the range observed in IBDpatients (n=17; R²=0.51; FIG. 2 a). We have found that neutrophilphagocytosis in whole blood is significantly reduced in Crohn's Diseasepatients, relative to healthy controls (see FIG. 2 b). This reductionwas intermediate between healthy controls and that observed in PAPpatients, and suggests that the elevated anti-GM-CSF antibodies observedin the Crohn's Disease patients do have a significant effect uponneutrophil phagocytosis.

We then asked whether GM-CSF bioactivity, as measured by CD11bactivation on neutrophils in whole blood, would also be reduced inCrohn's Disease patients. As shown in FIG. 3, we have found that GM-CSFdependent CD11b activation on neutrophils is significantly reduced inCrohn's Disease patients, relative to healthy controls.

Taken together, these studies have identified anti-GM-CSF as a novelregulator of neutrophil function and IBD phenotype. Further thesestudies establish the predictive value of the serum anti-GM-CSF antibodywith regard to IBD phenotype. The risk associated with the serumanti-GM-CSF antibody level with regard to IBD phenotype and behavior isat least comparable to that associated with CARD15 SNP carriage,suggesting a functional interaction. More importantly, as both GM-CSFadministration and GM-CSF neutralization are in development as therapiesfor inflammatory bowel diseases and other autoimmune disorders, thisraises the possibility that determination of the anti-GM-CSF level in anindividual patient may guide biologic therapy, in terms of whetheraugmentation or inhibition of GM-CSF bioactivity would be beneficial.

CD11b activity in response to exogenous GM-CSF may be used as abiomarker to classify subtypes of inflammatory bowel disease, whereinindividuals having reduced CD11b activity are more likely to have CD,and less likely to have UC. Thus, in this aspect of the presentinvention, determination of CD11b activity, particularly in response toGM-CSF, may be used as a diagnostic assay to classify subtypes ofinflammatory bowel disease, particularly ulcerative colitis and Crohn'sdisease. In one aspect, CD11b activity on monocytes is measured todistinguish between CD and UC patients. In another aspect, CD11bactivity on neutrophils is determined to distinguish between CD and UCpatients. Such classification of IBD patients permits targeted therapyand prediction of disease characteristics, behavior and likely outcome.

In yet another aspect, the instant invention provides a method ofperforming an overall risk assessment, comprising the steps of obtaininga serum sample from a individual suspected of having an IBD, determiningthe concentration of anti-GM-CSF relative to neutrophil function, IBDphenotype, CARD15 variants or commercial IBD serology to obtain a ratio,then using the determined ratio to determine whether the individual hasCrohn's disease or whether an individual having an IBD has a highlikelihood of disease progression and surgery. The commercial IBDserology includes, for example, ASCA, pANCA, CBir1, I2, or OmpCantibodies. The disclosure of U.S. Pat. No. 6,218,129 Walsh et al., U.S.Pat. No. 6,183,951, Plevy et al., and U.S. Pat. No. 5,932,429 Targan etal., describing methods of diagnosing IBD are all incorporated herein byreference.

In another aspect of the instant invention, the novel biomarkers andmethods disclosed herein may be used to predict or determineinflammatory bowel disease location and behavior via measurement ordetection of anti-GM-CSF antibody concentrations with or without thepresence of the CARD15 single nucleotide polymorphism.

Table 1 depicts data representing the interactions between the CARD15Crohn's Disease (CD) susceptibility gene and anti-GM-CSF antibody withrespect to disease location and behavior. In Table 1, the demographiccharacteristics and CD location for a cohort of pediatric onset IBDpatients are shown, together with the frequency of CARD15 SNP carriage,stricturing or penetrating behavior (B2/B3), and surgery. Serumanti-GM-CSF (mcg/ml) is given as the median (25th, 75th percentile), a,b, c, d, e=p<0.05 by Kruskal-Wallis test with Dunn's multiple comparisontest versus the indicated group. The patients have been followed for amedian (25th, 75th percentile) of 4 (2, 6) years from diagnosis. Thediagnosis of CD or UC is made based upon established endoscopic,histological, and radiographic criteria (Hanauer S. B. 2006). In thissystem, ileal involvement is classified as L1, colonic as L2, andileo-colonic as L3. CD phenotype is assigned as per the Montrealclassification, with patients with ileal or ileo-colonic disease groupedtogether as small bowel CD (CD_(SB)), and patients with colonic CDgrouped as CD_(C) (Satsangi J. 2006).

The individuals have been genotyped for the three predominant CARD15 CDsusceptibility SNPs (R702w, G908R and 1007fs). Carriage of at least oneSNP has been recorded as positive with respect to the current analysis.Serum anti-GM-CSF concentrations may be determined using an ELISA asdescribed in the Examples herein. Genotyping for the NOD2/CARD15variants associated with CD, R702W (SNP8), G908R (SNP12), and 1007fs(SNP13) is performed using the protocol published in Tomer G. 2003,incorporated herein by reference. Titers of the ASCA, OmpC, 12, andCBir1 antibodies which are associated with small bowel CD, and pANCA,which is associated with UC and colonic CD, may be determined by acommercial laboratory (Prometheus Laboratories, San Diego, Calif.).

In our studies, 40% to 45% of the CD individuals with ileal orileocolonic disease exhibited CARD15 SNP carriage and stricturing orpenetrating disease behavior over time (B2/B3), with 30% requiring atleast one surgery. Consistent with our prior results, we have confirmedthat serum anti-GM-CSF is up regulated in CD patients with ileal orileo-colonic involvement, relative to patients with Crohn's colitis, UC,or healthy controls.

TABLE 1 Anti-GM-CSF and Disease Location and Behavior CARD15 Anti-GM-CSFLocation n Age Male SNP (mcg/ml) B2/B3 Surgery L1: Ileal, 20 13 75% 45%2.2 (0.9, 6.7) 45% 30% a d, e L3: Ileo-colonic, 86 12 52% 47% 2.2 (0.7,9.4) 37% 33% b c, d, e L2: Colonic, 51 11 61% 16% 0.7 (0.4, 1.6) 14% 16%c b UC, d 43 12 60% 16% 0.7 (0.2, 1.3) N/A 16% a, b Control, e 20 13 70%Not done 0.4 (0.2, 0.6) N/A N/A a, b

Referring to Table 1, the mean (95% CI) anti-GM-CSF level was equal to10 (4, 16) mcg/ml in CD patients carrying at least one CARD15 SNPcompared to 9 (3, 15) mcg/ml in those without a CARD15 SNP. Similarly,47% of patients with elevated anti-GM-CSF (≧2 mcg/ml) carried a CARD15SNP, while 53% did not. This suggested that increased anti-GM-CSF wasnot simply a function of CARD15 SNP carriage.

The relative risk (RR) for ileal or ileo-colonic involvement (CD_(SB))in a CD patient carrying a CARD15 susceptibility SNP was equal to 5.2,while the RR for a CD patient with an anti-GM-CSF level ≧2 mcg/ml wasequal to 4, and the RR for a CD patient with both an anti-GM-CSF level≧2 mcg/ml and CARD15 SNP was equal to 10.2 (p<0.001 for each).Therefore, elevated anti-GM-CSF levels appear to interact with CARD15SNPs to increase susceptibility for CD_(SB). Importantly, anti-GM-CSFlevels did not change over one year in patients who had achievedclinical remission with anti-TNF therapy (2.4±0.8 mcg/ml vs 3.3±1.3mcg/ml, n=6), indicating that these are a stable feature of theirdisease, and do not simply reflect a response to mucosal inflammation.

The chi-square test of independence has found a significantly differentprevalence of L1/L3 (vs. L2), CARD15 mutation, and high anti-GM-CSF (≧2mcg/ml) in patients with CD compared to patients with UC (p-values<0.0001, 0.0155, 0.0032 respectively). Hence, we have investigated theassociation of anti-GM-CSF with the disease location, behavior andsurgery while controlling for CARD15 mutation, separately in patientswith CD and in patients with UC, the results of which are shown inTable 1. The median anti-GM-CSF level was increased two-fold in CDpatients with penetrating or stricturing behavior (B2/B3), compared tothose with inflammatory behavior (B1). After stratifying for individualswith ileal or ileo-colonic CD, the median anti-GM-CSF level was equal to1.8 (0.7, 7.7) in those who did not require surgery, versus 6.6 (0.7,10) in those who did require surgery (p=0.04). Consistent with this, theodds ratio (OR) for surgery in CD patients with elevated anti-GM-CSF wasequal to 3.2 (p<0.01).

Using the Cochran-Mantel-Haenszel (CMH) test controlling for CARD15 SNPcarriage, it was found that anti-GM-CSF level (high vs. low) issignificantly associated with the disease location distribution (L2 vs.L1/L3), the disease behavior distribution (B1 vs. B2/B3) and surgerystatus (p-values=0.0005, 0.0076, 0.0038 respectively). Morespecifically, for a CD patient with a high anti-GM-CSF level (≦2mcg/ml), the disease is less likely to be only colonic (L2) (RelativeRisk=0 with CARD15 mutation, 0.63 with no CARD15 mutation), less likelyto be non-stricturing, non-penetrating B1 (Relative Risk=0.7366), andmore likely treated with a surgery (Relative Risk=2.155), independent ofthe presence/absence of CARD 15 mutation. The mean elapsed time sincediagnosis is not significantly different in the patients with highanti-GM-CSF level than in the patients with a low anti-GM-CSF level.Therefore, these results indicate a significant effect of anti-GM-CSFlevel, independent of CARD15 mutation effect.

In contrast, using the CMH test anti-GM-CSF level (high vs. low)controlling for CARD 15 SNP status, is not significantly associated withthe surgery status in patients with UC.

As such, one aspect of the present invention provides kits and methodsof classifying IBD patients by determining the concentration ofanti-GM-CSF antibody in blood or serum samples from individualssuspected of having or diagnosed with IBD. In particular, anti-GM-CSFlevels may be used to distinguish CD patients with ileal or ileo-colonicdisease (in which surgery is a more likely outcome) from colonic CDpatients, UC patients, and normal (healthy) patients. By classifying theIBD patient into a particular subgroup, the appropriate course oftherapy may be determined, reducing the likelihood of administeringtreatments that will not be successful, thereby stemming diseaseprogression and improving the individual's overall quality of life.

For example, determination of anti-GM-CSF antibody levels in anindividual also be used to guide biologic therapy, in terms of whetheraugmentation or inhibition of GM-CSF bioactivity would be beneficial,comprising the steps of obtaining a sample from the individual,determining the concentration of serum anti-GM-CSF antibodyconcentration in the sample, wherein a mean serum anti-GM-CSF antibodyconcentration of greater than or equal to about 1 mcg/ml, or great thanor equal to about 1.5 mcg/ml, or greater than or equal to about 2.0mcg/ml, or within the range of about 1.0 mcg/ml to 2.5 mcg/ml indicatesan increased risk for disease progression and surgery. In patientshaving elevated risk of surgery, treatments such as anti-TNF alpha (suchas Humira or Remicade), or GM-CSF therapy would be indicated as thepreferred therapy. In this regard, the instant methods, and kitsembodying these methods, allow for improved treatment of individualslikely to require surgery.

Thus, the present invention is directed to a highly sensitive method ofdiagnosing, classifying, or characterizing disease in individuals havingan inflammatory bowel disease, such that disease progression and/or theappropriate course of treatment may be readily determined, and kitsutilizing this method.

A variety of assay formats can be used to determine anti-GM-CSF antibodylevels in a sample.

Flow cytometry can be used to determine anti-GM-CSF antibody levelsaccording to a method of the invention. Such flow cytometric assays,including bead based immunoassays, can be used to determine anti-GM-CSFantibody levels in the same manner as used to detect serum antibodies toCandida albicans and serum antibodies to HIV proteins (see, for example,Bishop and Davis, J. Immunol. Methods 210:79-87 (1997); McHugh et al.,J. Immunol. Methods 116:213 (1989); Scillian et al., Blood 73:2041(1989), each of which is incorporated by reference herein).

Phage display technology for expressing a recombinant antigen specificfor anti-GM-CSF antibodies also can be used to determine the level ofanti-GM-CSF antibody. Phage particles expressing the antigen specificfor anti-GM-CSF antibody, or an antigen specific for anti-GM-CSFantibody, can be anchored, if desired, to a multiwell plate using anantibody such as an antiphage monoclonal antibody (Felici et al.,“Phage-Displayed Peptides as Tools for Characterization of Human Sera”in Abelson (Ed.), Methods in Enzymol. 267, San Diego: Academic Press,Inc. (1996), which is incorporated by reference herein).

A variety of immunoassay formats including competitive andnon-competitive immunoassay formats also are useful the methods of theinvention (Self and Cook, Curr. Opin. Biotechnol. 7:60-65 (1996), whichis incorporated by reference). Immunoassays encompass capillaryelectrophoresis based immunoassays (CEIA) and can be automated, ifdesired. Immunoassays also can be used in conjunction with laser inducedfluorescence (see, for example, Schmalzing and Nashabeh, Electrophoresis18:2184-93 (1997)); Bao, J. Chromatogr. B. Biomed. Sci. 699:463-80(1997), each of which is incorporated herein by reference). Liposomeimmunoassays, such as flow-injection liposome immunoassays and liposomeimmunosensors, also can be used to determine anti-GM-CSF antibodyconcentration.

Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), canbe particularly useful in a method of the invention. An ELISA, forexample, can be useful for determining whether a sample is positive foranti-GM-CSF antibodies or for determining the anti-GM-CSF antibody levelin a sample. An enzyme such as horseradish peroxidase (HRP), alkalinephosphatase (AP), β-galactosidase or urease can be linked to a secondaryantibody selective for anti-GM-CSF antibody, or to a secondary antibodyselective for anti-GM-CSF antibody for use in a method of the invention.A horseradish-peroxidase detection system can be used, for example, withthe chromogenic substrate tetramethylbenzidine (TMB), which yields asoluble product in the presence of hydrogen peroxide that is detectableat 450 nm. An alkaline phosphatase detection system can be used with thechromogenic substrate p-nitrophenyl phosphate, for example, which yieldsa soluble product readily detectable at 405 nm. Similarly, aβ-galactosidase detection system can be used with the chromogenicsubstrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which yields asoluble product detectable at 410 nm, or a urease detection system canbe used with a substrate such as urea-bromocresol purple (SigmaImmunochemicals, St. Louis, Mo.). A useful secondary antibody linked toan enzyme can be obtained from a number of commercial sources; goatF(ab′)₂ anti-human IgG-alkaline phosphatase, for example, can bepurchased from Jackson Immuno-Research (West Grove, Pa.).

A radioimmunoassay also can be useful for determining the level ofanti-GM-CSF antibodies in a sample. A radioimmunoassay using, forexample, an iodine¹²⁵ labeled secondary antibody (Harlow and Lane,Antibodies A Laboratory Manual Cold Spring Harbor Laboratory: New York,1988, which is incorporated herein by reference) is encompassed withinthe invention.

A secondary antibody labeled with a chemiluminescent marker also can beuseful in the methods of the invention. Such a chemiluminescentsecondary antibody is convenient for sensitive, non-radioactivedetection of anti-GM-CSF antibodies and can be obtained commerciallyfrom various sources such as Amersham Lifesciences, Inc. (ArlingtonHeights, Ill.).

In addition, a detectable reagent labeled with a fluorochrome can beuseful in the methods of the invention for determining the levels ofanti-GM-C SF antibody in a sample. Appropriate fluorochromes include,for example, DAPI, fluorescein, Hoechst. 33258, R-phycocyanin,B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red or lissamine. Aparticularly useful fluorochrome is fluorescein or rhodamine. Secondaryantibodies linked to fluorochromes can be obtained commercially. Forexample, goat F(ab′)₂ anti-human IgG-FITC is available from TagoImmunologicals (Burlingame, Calif.).

A signal from the detectable reagent can be analyzed, for example, usinga spectrophotometer to detect color from a chromogenic substrate; aradiation counter to detect radiation, such as a gamma counter fordetection of iodine-125; or a fluorometer to detect fluorescence in thepresence of light of a certain wavelength. For detection ofenzyme-linked reagents, a quantitative analysis of the amount ofanti-GM-CSF antibody can be made using a spectrophotometer such as anEMAX Microplate Reader (Molecular Devices, Menlo Park, Calif.) inaccordance with the manufacturer's instructions. If desired, the assaysof the invention can be automated or performed robotically, and thesignal from multiple samples can be detected simultaneously.

Immunoassays using a secondary antibody selective for anti-GM-CSFantibodies are particularly useful in the methods of the invention. Asused herein, the term “antibody” means a population of immunoglobulinmolecules, which can be polyclonal or monoclonal and of any isotype. Asused herein, the term “antibody” encompasses an immunologically activefragment of an immunoglobulin molecule. Such an immunologically activefragment contains the heavy and light chain variable regions, which makeup the portion of the antibody molecule that specifically binds anantigen. For example, an immunologically active fragment of animmunoglobulin molecule known in the art as Fab, Fab′ or F(ab′)₂ isincluded within the meaning of the term antibody.

Using the above-described methods, an individual may be identified asbeing an optimal candidate for GM-CSF treatment. “GM-CSF” refers to aprotein that stimulates the production of granulocytes and macrophagesby stem cells. GM-CSF used in the practice of the invention includes anypharmaceutically safe and effective human GM-CSF (e.g., the human GM-CSFwith amino acid having the following sequence: Ala Pro Ala Arg Ser ProSer Pro Ser Thr Gln Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala Arg ArgLeu Leu Asn Leu Ser Arg Asp Thr Ala Ala Glu Met Asn Glu Thr Val Glu ValIle Ser Glu Met Phe Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu GluLeu Tyr Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu ThrMet Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser CysAla Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp Phe LeuLeu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu (SEQ ID NO:1)), orany derivative thereof having the biological activity of human GM-CSF.Derivatives of GM-CSF may be (i) one in which one or more of the aminoacid residues of the protein are substituted with a conserved ornon-conserved amino acid residue, and such substituted amino acidresidue may or may not be one encoded by the genetic code, (ii) one inwhich one or more of the amino acid residues of the protein include asubstituent group, (iii) one in which the mature protein is fused withanother compound, such as a compound to increase the half-life or thepolypeptide (for example, polyethyleneglycol), (iv) one in whichadditional amino acids are fused to the mature protein, such as a leaderor secretory sequence or a sequence which is employed for purificationof the mature protein, or (v) one in which the protein is fused with alarger protein, i.e., an antibody or Fc. Examples of GM-CSFs include,but are not limited to, native GM-CSF, molgramostim (bacteria-derivedGM-CSF), ragramostim (CHO-derived GM-CSF), sargramostim(Leukine—yeast-derived GM-CSF), and pegylated GMCSF (i.e., pegylatednative GM-CSF or pegylated GMCSF derived from any source).

Recombinant GM-CSF may be used with the methods of the instantinvention. “Recombinant GM-CSF” refers to either to GM-CSF synthesizedin a cell into which a nucleic acid encoding exogenous GM-CSF has beenintroduced, or a cell in which the endogenous GM-CSF gene has beenstimulated to overproduce GM-CSF by the introduction of regulatoryelements that induce a high rate of transcription of the endogenousGM-CSF gene.

The GM-CSF used may be recombinant human GM-CSF (rhu GM-CSF), such asLeukine®. (Berlex Inc., Bothell, Wash.). Leukine®. (generically termed“sargramostim”) is a biosynthetic, yeast-derived, recombinant humanGM-CSF, consisting of a single 127 amino acid glycoprotein that differsfrom the endogenous human GM-CSF shown having the sequence as set forthabove in which arginine is substituted with leucine at position 23.Leukine® is produced in the yeast Saccharomyces cerevisiae.

Leukine® has been shown to exhibit the same hematopoietic effects asthose induced by endogenous GM-CSF, namely, the stimulation ofprogenitor cells committed along the granulocyte-macrophage pathway toform neutrophils, monocytes, macrophages, and eosinophils (TechnicalProduct Report: Leukine® Liquid, Immunex Corporation, Seattle, Wash.,1997, which is herein incorporated by reference). Leukine®, likeendogenous GM-CSF, also promotes the differentiation of progenitor cellsgiving rise to erythrocytes and megakaryocytes. In addition tostimulating hematopoiesis, Leukine® enhances many of the functionalactivities of mature neutrophils, monocytes and macrophages, such aschemotaxis, growth factor secretion, anti-tumor activity, antibacterialand antifungal activities, and so on.

Leukine® Liquid is a sterile injectable aqueous solution generally soldin 1 ml vials containing 500 μg/ml (2.8×10⁶ IU) sargramostim; 40 mg/mlmannitol; 10 mg/ml sucrose; 1.2 mg/ml tromethamine; sterile water; and1.15% benzyl alcohol. LEUKINE® Lyohphilized is also sold, and typicallyis packaged in vials containing a sterile lyophilized powder forreconstitution with 1 ml sterile water. LEUKINE® Lyophilized may contain250 μg or 500 μg sargramostim (1.4 or 2.8×10⁶ IU); 40 mg mannitol; 10 mgsucrose; and 1.2 mg tromethamine. LEUKINE® Liquid and reconstitutedsolutions of LEUKINE® Lyophilized are stored refrigerated at 2-8° C.

Typically, the aqueous solution of GM-CSF is administered bysubcutaneous injection or intravenous infusion. However, other methodssuch as oral, intraperitoneal, subdermal, and intramuscularadministrations may be used. Doses delivered may be the same as thosedelivered to stimulate an immune response in humans for other diseasepurposes. In certain embodiments, doses may be about 100 to about 1500μg (including any values therebetween, such as about 250, 500, and 1000μg) once per week when administered via subcutaneous injection.

The methods of administration of GM-CSF as described in US 2007/0041938,Pettit, et al. are incorporated herein by reference.

In addition, using the above-described methods, patients may beidentified that would benefit from administration of anti-TNF. Thistherapy is sold under the trade name Humira®, and is described in U.S.Pat. No. 6,090,382, Salfeld et al., incorporated herein by reference.This therapy is administered using standard methods as known to one ofordinary skill in the art.

The following examples are intended to illustrate but not limit thepresent invention.

Example I Determining Anti-GM-CSF Antibody Activity for Classificationof Subtypes

GM-CSF concentration in whole blood or serum samples obtained from apatient suspected of having an inflammatory bowel disease is determinedusing the methods as described in Uchida, et al. Blood, 2004,incorporated herein by reference, and described herein.

Anti-GM-CSF antibodies are assayed using serum samples that are eitherimmediately obtained or previously collected and stored at—70° C. untilanalysis. Anti-GM-CSF antibody concentration of a serum sample from anindividual of interest is then measured. The serum sample is diluted1:100, 1:1000 and 1:3000 with phosphate buffered saline (PBS) containing1% bovine serum albumin and 0.1% Tween 20. Separate 50 μl aliquots ofdiluted serum and the affinity-purified anti-GM-CSF antibody isolatedfrom the serum of patients with pulmonary alveolar proteinosis as astandard (0-50 ng/ml) were incubated at room temperature for 40 minutesin ELISA plates previously coated overnight at 4° C. with 1 μg/mlrhGM-CSF (Leukine® Berlex) and blocked for 1 hour with StabilCoat®(SurModics). After washing five times with PBS 0.1% Tween 20 (PBST),anti-GM-CSF antibody captured by rhGM-CSF is detected by peroxidaselabeled anti-human IgG F(ab)₂ antibody (Sigma). Tetramethylbenzidine isused as a substrate and absorbance is measured at 450 nm after stoppingthe reaction with 1N sulfuric acid. Assays are performed in duplicateand the mean of the two results used.

Example II Anti-GM-CSF Concentration as a Predictor of Disease Behavior

The methods of Example 1 are carried out essentially as described above.Following performance of the above-described methods, the valuesobtained from the assay are then used to classify the individual aseither having colonic, or ileal or ileo-colonic. Values greater than orequal to about 2 mcg/ml indicate that the disease behavior is lesslikely to be only colonic (L2), less likely to be non-stricuring,non-penetrating (B1), and more likely to be treated with a surgery.Assessment of the presence or absence of the CARD 15 mutation is notnecessary for this determination.

Example III Determining CD11b Stimulation Index for Classification ofSubtypes

The CD11b Stimulation Index on neutrophils from whole blood samplesobtained from an individual suspected of having an inflammatory boweldisease is determined using the methods as described in Uchida, et al.NEJM, 2007, incorporated herein by reference, and described herein.Briefly, this assay is performed using whole blood samples from IBDpatients or healthy controls in the absence or presence of stimulationwith exogenously added GM-CSF and then cell surface CD11b is quantifiedon neutrophils (and/or also on monocytes and eosinophils) is thenmeasured by flow cytometry. Neutrophils can be identified with the highexpression level of CD16 determined by fluorescein isothiocyanate(FITC)-conjugated anti-CD16 antibody. Monocytes can be determinedAPC-conjugated anti-CD14 antibody. Eosinophils can be determinedmoderate expression of CD16 and specific cell size and complexity. Thisassay provides a method to quantify the level of neutrophil (as well asmonocyte and eosinophil) innate immune function in IBD patients.

The phycoerythrin (PE)-conjugated monoclonal antibodies against CD11b ispurchased from BD Biosciences (San José, Calif, USA). CD11b modulatesthe adherence of polymorphonuclear neutrophils and monocytes tofibrinogen and positive ICAM-1 endothelia.

Preparation for Flow-Cytometry Analyses

Heparinized whole blood is incubated for 30 minutes in the presence orabsence of 10 ng of human GM-CSF per milliliter (Leukine, Berlex) or 10ng of mouse GM-CSF per milliliter (R&D Systems) and incubated for 30minutes. The samples (50 μL) are then mixed with 50 μL PBS inpolystyrene round-bottom tubes (5 mL Falcon, BD-Pharmingen, Heidelberg,Germany) and incubated with incubated with 5 μl, undiluted FITC-, APC-and PE-conjugated antibodies for 30 minutes on ice in the dark. Thereaction is stopped and cells are fixed by washing the suspension with 3ml of BD FACS Lysing solution (BD Biosciences).

Flow cytometry is performed with a FACS Calibur flow cytometer (BDBiosciences) equipped with a 15-mW argon laser emitting at 488 nm. Thefluorescence signals of FITC and PE are detected with 530/30 nm(channel 1) and 585/42 nm band pass filters (channel 2) with correctionof the spectral overlap by color compensation. Calibration of the flowcytometer is performed using standard fluorescent microbeads (CaliBRITE,BD Biosciences). Analysis of the fluorescence properties of 50,000events is performed using CellQuest software (BD Biosciences). Theincrease in CD11b levels after GM-CSF priming (CD11b stimulation index)is calculated as the mean fluorescence intensity of CD11b primed byGM-CSF minus that of CD11b on nonprimed samples, divided by the meanfluorescence intensity of nonprimed samples and multiplied by 100.

GM-CSF stimulation of CD11b on neutrophils (and/or monocytes oreosinophils) is (are) compared to the corresponding cell types in acontrol group (such as normal, healthy controls) or known standards suchas patients diagnosed with small bowel Crohn's disease or ulcerativecolitis. A determination that reduced or absent GM-CSF-mediated increasein cell surface CD11b levels in a sample is one mechanism to confirmthat the increased level of GM-CSF autoantibodies have functionalsignificance with respect to GM-CSF bioactivity in a given IBD patient.

The foregoing description of examples and embodiments of the subjectapplication has been presented for purposes of illustration anddescription. It is not intended to be exhaustive or to limit the subjectapplication to the precise form disclosed. Obvious modifications orvariations are possible in light of the above teachings. The embodimentswere chosen and described to provide the best illustration of theprinciples of the subject application and its practical application tothereby enable one of ordinary skill in the art to use the subjectapplication in various embodiments and with various modifications assuited to the particular use contemplated. All such modifications andvariations are within the scope of the subject application as determinedby the appended claims when interpreted in accordance with the breadthto which they are fairly, legally and equitably entitled.

1. A diagnostic kit for the diagnosis and prognosis of inflammatorybowel diseases in a mammalian subject comprising: a probe specific forGM-CSF wherein the probe is capable of detecting a concentration ofanti-GM-CSF antibodies, such that a diagnosis or prognosis of thesubject may be made; and reactants for detecting the concentration ofanti-GM-CSF antibodies.
 2. The diagnostic kit according to claim 1,wherein the reactants for detecting the concentration of anti-GM-CSFantibodies function in a method selected from the group consisting of insitu hybridization, hybridization, and recognition by marked specificantibodies, the method being conducted on filter, on solid support, insolution, or on gel, by using at least one technique selected from thegroup consisting of a sandwich method, Dot blot hybridization, isotopicor non-isotopic labeling, cold probe techniques, double immunodiffusion,counter-immunoelectrophoresis, and hemagglutination.
 3. The diagnostickit according to claim 1, wherein the probe is an antigen reactive toanti-GM-CSF antibodies.
 4. The diagnostic kit according to claim 1,wherein the probe is immobilized on a solid support.
 5. The diagnostickit according to claim 1, wherein the antigen forms an antigen-antibodycomplex with the anti-GM-CSF antibodies.
 6. The diagnostic kit accordingto claim 1, wherein the detection reactants comprise a reporter groupconjugated to a binding agent.
 7. The diagnostic kit according to claim1, wherein the binding agent is selected from the group consisting ofanti-immunoglobulins, Protein G, Protein A and lectins.
 8. Thediagnostic kit according to claim 1, wherein the reporter group isselected from the group consisting of radioisotopes, fluorescent groups,luminescent groups, enzymes, biotin and dye particles.
 9. The diagnostickit according to claim 1, wherein the probe is a phage particleexpressing an antigen specific for anti-GM-CSF antibody.
 10. A methodfor the diagnosis and prognosis of inflammatory bowel diseases in amammalian subject comprising the following steps: (a) obtaining abiological sample from an individual suspected of having an inflammatorybowel disease; (b) determining the concentration of anti-GM-CSFantibodies in the sample; and (c) correlating the concentration ofanti-GM-CSF antibodies in the sample to known standards.
 11. The methodaccording to claim 10, wherein the inflammatory bowel disease is apathology selected from the group consisting of small bowel Crohn'sdisease (CD_(SB)), Colonic Crohns disease (CD_(C)), and UlcerativeColitis (UC).
 12. The method according to claim 10, wherein thebiological fluid is serum.
 13. The method according to claim 10, furthercomprising correlating the concentration of anti-GM-CSF antibodies inthe sample to known standards to predict the severity of theinflammatory bowel disease.
 14. The method according to claim 10,further comprising correlating the concentration of anti-GM-CSFantibodies in the sample to known standards to predict whether thesubject will require surgery.
 15. The method according to claim 10,further comprising correlating the concentration of anti-GM-CSFantibodies in the sample to known standards to select the appropriatetherapeutic treatment for the inflammatory bowel disease, whereinelevated anti-GM-CSF antibodies indicate that the patient is a candidatefor therapies selected from anti-TNFα therapy, GM-CSF administration, orcombinations thereof.
 16. The method according to claim 10, whereincorrelating the concentration of anti-GM-CSF antibodies in the sample toknown standards is used to distinguish between pathologies selected fromthe group consisting of small bowel Crohn's disease (CD_(SB)), ColonicCrohns disease (CD_(C)), and Ulcerative Colitis (UC).
 17. The methodaccording to claim 10, further comprising the step of a determination ofGM-CSF dependent up-regulation of cell surface CD11b in cells of thesample.
 18. The method according to claim 10, wherein determination ofGM-CSF dependent up-regulation of cell surface CD11b in cells of thesample is used as a diagnostic assay to distinguish between UC and CDpatients.
 19. The method according to claim 10, wherein theconcentration of anti-GM-CSF antibodies in the sample is determinedusing at least a probe specific for GM-CSF wherein the probe is capableof detecting a concentration of anti-GM-CSF antibodies, such that adiagnosis or prognosis of the individual may be made.
 20. The methodaccording to claim 10, wherein the concentration of anti-GM-CSFantibodies in the sample is determined by providing an immobilizedantigen reactive to anti-GM-CSF antibodies, contacting the sample withthe antigen to form an antigen-antibody complex, washing the complex toremove non-specifically bound components, followed by detecting thecaptured anti-GM-CSF antibodies.
 21. A method for the diagnosis andprognosis of inflammatory bowel diseases in a mammalian subjectcomprising the following steps: (a) obtaining a biological sample froman individual suspected of having an inflammatory bowel disease; (b)determining the concentration of anti-GM-CSF antibodies in the samplerelative to neutrophil function, IBD phenotype, CARD15 variants orcommercial inflammatory bowel disease serology; (c) correlating theconcentration of anti-GM-CSF antibodies in the sample to known standardsand (d) diagnosing the individual based on these results.
 22. A methodfor the diagnosis of irritable bowel syndrome, the method comprising:obtaining a blood or serum sample from a subject presenting withsymptoms common to inflammatory bowel disease and irritable bowelsyndrome; and determining whether the sample contains an elevated levelof anti-GM-CSF antibodies, wherein if the sample contains an elevatedlevel of anti-GM-CSF antibodies, a diagnosis of inflammatory boweldisease is substantially concluded.
 23. The method according to claim22, wherein the anti-GM-CSF antibodies are qualitatively determined. 24.The method according to claim 22, wherein the step of determiningwhether the sample contains an elevated level of anti-GM-CSF antibodiesincludes contacting the sample with immobilized polyclonal antibodies tohuman GM-CSF to create an antibody bound sample.
 25. A diagnostic assayfor determining whether a blood or serum sample contains an elevatedlevel of anti-GM-CSF antibodies as compared to a reference value forhealthy control subjects, the assay comprising: obtaining a human bloodor serum sample from a person presenting with symptoms common betweeninflammatory bowel disease and irritable bowel syndrome; contacting thesample with immobilized polyclonal antibodies to anti-GM-CSF antibodiesto create an antibody bound sample; contacting the treated sample withenzyme-linked polyclonal antibodies such that the enzyme-linkedpolyclonal antibodies are allowed to bind to capture human GM-CSF tocreate an enzyme-linked antibody bound sample; and determining whetherthe enzyme-linked antibody bound sample contains an elevated level ofanti-GM-CSF antibodies as compared to a reference value for healthycontrol subjects, wherein if the enzyme-linked antibody bound samplecontains an elevated level of anti-GM-CSF antibodies, a diagnosis ofinflammatory bowel disease is substantially concluded.
 26. Thediagnostic assay according to claim 22, wherein the assay comprises anenzyme-linked immunoassay.
 27. A method for the diagnosis and prognosisof inflammatory bowel diseases in a mammalian subject comprising thefollowing steps: (a) obtaining a biological sample from an individualsuspected of having an inflammatory bowel disease; (b) determining theconcentration of a biological marker, the marker being a cell-surfaceadhesion molecule present on myeloid cells or CD11b in the sample; and(c) correlating the concentration of marker in the sample to knownstandards.
 28. The method according to claim 27, wherein theconcentration of a biological marker is determined by GM-CSF priming,which increases cell-surface levels of CD11b on neutrophils contained inthe sample.
 29. The method according to claim 27, wherein GM-CSFautoantibodies block the GM-CSF-stimulated increase in cell surfaceCD11b levels.
 30. The method according to claim 27, wherein the additionof exogenous GM-CSF to whole blood from the subject is used to measurethe ability to stimulate a change in neutrophil CD11b levels.
 31. (b)placing the biological fluid in contact with at least either: (i) abiological marker obtained from a mammalian cell, the marker being acell-surface adhesion molecule present on myeloid cells or CD11b or (ii)an anti-marker antibody or antigen-binding portion thereof specific forthe CD11b.
 32. A method for the diagnosis and prognosis of inflammatorybowel diseases in a mammalian subject comprising the following steps:(a) obtaining a biological sample from an individual suspected of havingan inflammatory bowel disease; (b) determining the concentration of abiological marker, the marker being a cell-surface adhesion moleculepresent on myeloid cells or CD11b in the sample; and (c) correlating theconcentration of marker in the sample to known standards.
 33. A methodfor the diagnosis and prognosis of inflammatory bowel diseases in amammalian subject comprising the following steps: (a) obtaining abiological fluid from a subject suspected of having an inflammatorybowel disease; (b) placing the biological fluid in contact with at leasteither: (i) a marker obtained from an animal cell, the marker being acell-surface adhesion molecule present on myeloid cells or CD11b or (ii)an anti-marker antibody or antigen-binding portion thereof specific forin order to obtain either a biological binding in vitro between theantibody present in the biological fluid and the marker, or acompetitive immunological binding in vitro between the antibody presentin the biological fluid and the anti-marker antibody or theantigen-binding portion thereof specific for the CD11b; (c) detectingbinding obtained; and (d) correlating the binding antibodies in thesample to known standards.
 34. The method according to claim 33, whereinthe inflammatory bowel disease is a pathology selected from the groupconsisting of small bowel Crohn's disease (CD_(SB)), Colonic Crohn'sdisease (CD_(C)), and Ulcerative Colitis (UC).
 35. The method accordingto claim 33, wherein the biological fluid is serum.
 36. The methodaccording to claim 33, further comprising correlating the binding of thebiomarker in the sample to known standards to predict the severity ofthe inflammatory bowel disease.
 37. The method according to claim 33,further comprising correlating the binding of the biomarker in thesample to known standards to predict whether the subject will requiresurgery.
 38. The method according to claim 33, further comprising thebinding of the biomarker in the sample to known standards to select theappropriate therapeutic treatment for the inflammatory bowel disease,wherein elevated binding of the biomarker indicates that the patient isa candidate for therapies selected from anti-TNFα therapy, GM-CSFadministration, or combinations thereof.
 39. The method according toclaim 33, wherein correlating the binding of the biomarker in the sampleto known standards is used to distinguish between pathologies selectedfrom the group consisting of small bowel Crohn's disease (CD_(SB)),Colonic Crohns disease (CD_(C)), and Ulcerative Colitis (UC).
 40. Themethod according to claim 10, wherein if the subject exhibits low levelsof anti-GM-CSF antibody levels determined, relative to patients withcolonic CD and healthy controls, the subject is diagnosed withUlcerative Colitis (UC).
 41. The method according to claim 10, whereinif the subject exhibits high levels of anti-GM-CSF antibody levelsdetermined, relative to patients with colonic CD and healthy controls,the subject is diagnosed with small bowel CD (CD_(SB)).
 42. The methodaccording to claim 21, wherein the commercial IBD serology includesantibodies selected from the group consisting of ASCA, pANCA, CBir1, I2,OmpC or mixtures thereof.
 43. The method according to claim 27, whereinthe CD11b activity in response to exogenous GM-CSF may be used as abiomarker to classify subtypes of inflammatory bowel disease.
 44. Themethod according to claim 43, wherein individuals having reduced CD11bactivity are more likely to have CD, and less likely to have UC.
 45. Themethod according to claim 27, wherein the CD11b activity on monocytes ismeasured to distinguish between CD and UC patients.
 46. The methodaccording to claim 27, wherein the CD11b activity on neutrophils isdetermined to distinguish between CD and UC patients.